Interleukin-12 enhances the sensitivity of human osteosarcoma cells to 4-hydroperoxycyclophosphamide by a mechanism involving the Fas/Fas-ligand pathway.

Cyclophosphamide (CY) and its derivative ifosfamide are alkylating agents used to treat osteosarcoma (OS). The purpose of these studies was to determine whether alkylating agents affect the expression of Fas ligand (FasL) and whether interleukin 12 enhances the sensitivity of human OS cells to alkylating agents. 4-Hydroperoxycyclophosphamide (4-HC), the preactivated CY compound, and 4-hydroperoxydidechlorocloclophosphamide (4-HDC), its nonalkylating analogue, human OS LM6 cells, and a clone of cells derived by transfection with the interleukin 12 gene (LM6-#6) were used for these studies. Incubation of LM6 and LM6-#6 with 10 micro M 4-HC increased the expression of FasL mRNA (2.5- and 3.0-fold, respectively). By contrast, 4-HDC, Adriamycin (ADR), cisplatin (CDP), and methotrexate (MTX) had no effect on FasL mRNA expression. Increased FasL expression after treatment with 4-HC was also demonstrated by immunohistochemistry and flow cytometry. Drug-induced FasL was functional and mediated cell death. We examined the effect of FasL up-regulation by 4-HC on LM6 and LM6-#6 cells. Flow cytometry showed that LM6-#6 cells expressed 2.2-fold more Fas than LM6 cells. Cytotoxicity of 4-HC, 4-HDC, ADR, CDP, and MTX on LM6, LM6-neo, and LM6-#6 were quantified. Colony-forming assay revealed an IC(50) of 2.10 micro M for 4-HC in LM6-neo cells compared with 0.41 micro M in LM6-#6 cells. The IC(50) for 4-HDC, ADR, CDP, and MTX were not significantly different between the two cell lines. We concluded that the increased expression of Fas enhanced LM6-#6 sensitivity to 4-HC. These data indicate that Fas/FasL may be involved in the cytotoxic pathway of CY. Combining biological agents with chemotherapeutic agents that have complementary Fas/FasL pathway actions may offer new therapeutic alternatives.     

Overexpressed eIF4E is functionally active in surgical margins of head and neck cancer patients via activation of the Akt/mammalian target of rapamycin pathway.

PURPOSE: Overexpression of eIF4E in surgical margins of head and neck cancer patients is an independent risk factor for recurrence. We hypothesize that overexpressed eIF4E is functionally active in tumor margins through activation of the Akt/mammalian target of rapamycin (mTOR) pathway EXPERIMENTAL DESIGN: Western blots and/or immunohistochemistry were performed to determine whether phosphorylation of mTOR and activation of its downstream molecules eIF4E-binding protein-1 (4E-BP1) and p70 S6 kinase and the upstream modulator of mTOR, Akt, were expressed in margins overexpressing eIF4E. RESULTS: There was a significant association between phospho-4E-BP1 and eIF4E expression of a margin or a significant difference in phospho-4E-BP1 expression between the eIF4E-positive and -negative margins (P < 0.01). A significant association between eIF4E and phospho-p70 S6 kinase as well as eIF4E and phospho-mTOR was also noted (P < 0.05). Western blot analysis indicated a highly significant difference in the phosphorylation status of 4E-BP1 between tumors and resection margins. A total of 89% of the 4E-BP1-expressing margins expressed more of the phosphorylated (beta, gamma, and delta) isoforms, whereas 81% of the 4E-BP1-expressing tumors expressed more of the unphosphorylated alpha isoform. A similar difference in Akt activation was noted between eIF4E-positive margins and tumors (P < 0.05). CONCLUSIONS: Overexpression of eIF4E is functionally active in tumor margins through activation of the Akt/mTOR signaling pathway. The greater degree of expression of downstream targets and upstream regulators of mTOR in margins compared with the tumors indicates preferential activation of the Akt/mTOR signaling pathway in margins overexpressing eIF4E. Rapamycin analogs can potentially be used as adjuvant therapy for patients with eIF4E-positive margins.     

Porcine stem cell engraftment and seeding of murine thymus with class II+ cells in mice expressing porcine cytokines: toward tolerance induction across discordant xenogeneic barriers

BACKGROUND: Mixed hematopoietic chimerism is a reliable means of tolerance induction, but its utility has not been demonstrated in discordant xenogeneic combinations because of the difficulty in achieving lasting hematopoietic engraftment. Miniature swine are likely to be suitable organ donors for humans. To evaluate the ability of mixed chimerism to induce swine-specific tolerance in widely disparate xenogeneic recipients, this study aimed to achieve long-lasting chimerism in a pig to mouse combination. METHODS: Immunodeficient transgenic mice were developed by crossing transgenic founders carrying porcine interleukin-3, granulocyte macrophage-colony stimulating factor, and stem cell factor genes with severe combined immunodeficient mice or non-obese diabetic/severe combined immunodeficient mice. Swine bone marrow transplantation was performed in these mice, and porcine chimerism was followed for 20 weeks. RESULTS: Whereas swine cells became undetectable in all non-Tg littermates by 7 weeks, high levels of porcine hematopoietic chimerism, including the presence of porcine class II+ cells in the host thymus were maintained in Tg mice for >20 weeks. Colony-forming assays revealed the presence of large numbers of swine hematopoietic progenitor cells in the marrow of these mice at 20 weeks after bone marrow transplantation. CONCLUSIONS: These transgenic mice demonstrate for the first time that spontaneous migration of marrow donor antigen-presenting cells to an intact recipient thymus can occur and that porcine stem cells can persist in this highly disparate species combination. These data therefore support the feasibility of the eventual goal of tolerance induction by mixed chimerism in discordant xenogeneic combinations.     

Morphologic changes in efferent ductules and epididymis in estrogen receptor-alpha knockout mice

Estrogen has been shown to have an important role in fluid reabsorption in efferent ductules of the testis. Our previous study of the estrogen receptor-alpha knockout mouse (ERKO) showed that the efferent ductules and rete testis were primary targets of estrogen receptor function. In the present study, a more comprehensive evaluation of the ERKO male reproductive tract was performed to determine the severity of effects in efferent ductules as well as the epididymis. The following observations were found in ERKO males: 1) blind-ending efferent ductules were more prevalent in ERKO than in wild type (WT) tissues; 2) glycogen-containing cells were observed at the rete testis-efferent ductule junction; 3) the tubular diameters of the efferent ductules and initial segment epididymides were dilated; 4) efferent ductules were dilated between 130 to 300% over wild type ductules; 5) efferent ductule epithelial height was reduced nearly 50%; 6) microvilli of nonciliated cells of efferent ductules were 64% shorter in length; 7) cilia were reduced in number; 8) initial segment epithelium was displaced into regions adjacent to the rete testis and in short segments of the common region of efferent ductule; 9) apical, narrow, and clear cells of the epididymis also were abnormal in some regions; 10) in the corpus and cauda regions, sperm granulomas were noted in one third of the ERKO males. In conclusion, the entire reproductive tract is affected in ERKO males. The cells showing the greatest effects were estrogen receptor-positive cells. It appears that in the ERKO mouse there are developmental anomalies that must be considered separately from adult dysfunctional changes in the male reproductive tract.     

The treatment of premature arrest of growth plate with a novel engineered growth plate: experimental studies

OBJECTIVES: To observe the role of the engineered growth plate (EGP) in the treatment of premature arrest of growth plate and to establish a novel treatment method for the premature arrest of the growth plate. METHODS: The engineered growth plates were cultured for the first time by using polylactic acid (PLA) as cell scaffold seeded with growth plate chondrocytes and they were implanted into the medial proximal defects of growth plates of New Zealand rabbit tibia. The degree of deformity of the tibia was evaluated by X-ray and the expression of collagen II mRNA of regenerating growth plate was detected by in situ hybridization technique. RESULTS: Little deformity appeared in the EGP group 8 weeks after operation. Some deformity was seen in the EGP group 16 weeks after operation, whereas it's degree was much less than that of the control group (P = 0.0001). The degree of the angular deformity of the EGP precultured with bFGF and TGF-beta group was less than that of the EGP group (P < 0.05). The cells in the regenerating growth plate arranged in column and were stained blue by in situ hybridization technique. CONCLUSION: The EGP can prevent the formation of bony bridge and restore the growth of the damaged growth plate.     

糖皮质激素诱导性股骨头坏死模型的血管改变

目的　探讨在糖皮质激素诱导股骨头坏死过程中血管形态的改变与髓腔内脂肪填塞的关系。　方法　 12～ 18个月龄的健康日本大耳白兔 5 2只 ,按 2 5mg·kg-1·d-1注射地塞米松建立股骨头坏死模型 ,通过血管铸型扫描电镜观察、墨汁灌注切片光镜观察及图像分析等方法观察用药中和停药后股骨头病理和血管形态的变化。　结果　用药 2周出现股骨头髓腔内脂肪填塞 ,窦状隙血管因脂肪细胞压迫变细 ,血管铸型、墨汁灌注切片显示了血管表面压迹。病变随用药时间的延长而加重 ,窦状隙血管逐渐失去其结构特征。图像分析示股骨头内血管面积进行性下降 ,8周约为对照组的1/ 4。停药 6周负重区髓腔内仍存在明显脂肪化 ,血管纤细、稀少。实验 4周 ,股骨头内出现典型骨坏死灶。随时间的延长 ,坏死灶增大、股骨头坏死程度加重。　结论　髓腔内脂肪填塞是导致糖皮质激素诱导性股骨头坏死中缺血损害的重要病理因素之一 ,尤其在股骨头坏死的早期起着非常重要的作用     

胃底贲门癌根治性切除与扩大根治性切除的疗效对比

目的 探讨胃底贲门癌根治手术的最佳范围。方法 对４１８例胃底贲门癌患者施行根治性手术,其中扩大根治性切除１９２例（扩大组）,Ⅰ期１１例,Ⅱ期４０例,Ⅲ期１２１例,Ⅳ期２０例,根治性切除２２６例,Ⅰ期１９例,Ⅱ期５３例,Ⅲ期１３１,Ⅳ期２３例。对２且２术后５、１０年生存率进行对照分析。结果 ２种术式的５、１０年生存率;Ⅰ、Ⅱ期患者相似,差异无显著性意义;而Ⅲ期患者扩大组５、１０年生存率较根治组分别提     

病血糖难控因素中医药治疗

2型糖尿病(DM)患者血糖控制良好,可以大大减少或延缓并发症的出现,这已为多个国际糖尿病协作中心的观察所证实。然而,临床常遇到一些病人,虽药物剂量和种类不断调整,血糖仍然不能控制,除了常见的药物因素(如继发性磺脲类失效等)、饮食因素(如饮食控制不严格或结构不合理等)、运动因素(如疾病等原因致运动量不足)以外,尚可找到一些严重干扰降糖的诱因,我们把这些诱因称之为“血糖难控因素”。一旦找到,给予恰当的针对性治疗     

川红花产地环境及品质特性研究

川红花是传统的活血化瘀中药材,本文从川红花道地产区简阳的产地生态环境、产区的种植历史、与其它品种质量的比较等多方面研究了川红花的品质特性。结果表明,简阳具有悠久的川红花种植历史,良好的种植技术和品牌效应,产地生态环境因子（大气、土壤、农田灌溉、水质）符合中药材生产质量管理规范（GAP）要求;川红花品种的有效成份黄色素和红色素含量高,其吸收度分别为0.614和0.844,符合《中国药典》2000年版一部要求,并高于其它同生态条件种植的非地道性品种,其水溶性黄酮苷类的含量也高于其它品种;在不影响黄色素和黄酮苷类含量的条件下改进采收加工技术可大幅度提高红色素的含量,吸收度可达1.271,对红色素工业化提取极有利用价值;川红花药材的主要重金属和农药残留量均明显低于国家药材进出口绿色行业标准。实施川红花规范化栽培及按GAP要求建立生产基地后将进一步提高川红花的品质,增强川红花的国际市场竟争能力。